Abi 3500xl genetic analyser

The complete nucleoprotein gene of challenge virus standard (CVS-11) was amplified using primers Lys001 (5’-ACGCTTAACGAMAAA-3’) and 304 (5’-TTGACAAAATCTTCTCAT-3’) as described previously [33 (link)]. Amplification products were purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research) followed by cloning using the pGEM-T Easy vector system (Promega) according to the manufacturer’s instructions. Recombinant clones were characterized by sequencing using the BigDye Terminator V3.1 cycle sequencing kit (Thermo Fisher Scientific) and an ABI3500xL genetic analyser (Applied Biosystems) to determine orientation. A single recombinant clone containing the insert in the correct orientation with regard to the SP6 promoter was selected, and the insert was in vitro transcribed using the MegaScript kit (Ambion) according to the manufacturer’s instructions. In vitro transcribed RNA was purified using the RNA Clean and Concentrator-25 kit (Zymo Research) and quantified using the Qubit 3.0 fluorometer (Invitrogen). Contamination with plasmid DNA was ruled out with no-RT controls.

Larvae that were collected from the subintimal surface of the thoracic aorta as well as from peri-aortic nodules were submitted in 95% ethanol to the Department of Genetics, University of Pretoria, for genotyping. Previously published protocols were used for the deoxyribonucleic acid (DNA) extraction, polymerase chain reaction (PCR) amplification and sequencing [11 (link)]. Polymorphic forward primers of nine microsatellite loci for S. lupi from domestic dogs were labeled fluorescently. After combining the nine primer sets into a single multiplex reaction, the Quantitect Multiplex PCR kit (Qiagen) was used according to the manufacturer’s protocol. An ABI3500xl Genetic Analyser (Applied Biosystems), using GeneScan LIZ500 Internal Size Standard (Applied Biosystems) was used to separate and measure all allele sizes. By evaluating chromatograms in GeneMarker™ version 2.4.0. (SoftGenetics LLC), the genotype of each individual larva was established. Following published protocols, using the program Arlequin version 3.5.1, pairwise F_ST values were calculated from larvae collected from jackals and from dogs from three different provinces in South Africa [20 ].

The internal size standard was used during CE detection, which is crucial for accurate results of the CE platform. T500 (PEOPLESPOTINC, Beijing, China), which included 19 dye-labeled (“Orange”) DNA fragments (65, 70, 80, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 360, 390, 420, 450, 490, and 500 bp) and was selected as the internal size standard for calculating the fragment sizes of PCR products.
For CE progress, the PCR products were subsequently analyzed by adding 1 μL of each amplified product into 9 μL of a 17:1 mixture of Hi-Di formamide (Applied Biosystems, Foster City, CA, United States) and the T500 size standard. The mixture was denatured by heating at 95°C for 3 min and cooling at 4°C for 3 min. Samples were injected electrokinetically at 1.5 kV for 24 s and separated at 15 kV for 1,210 s by a run temperature of 60°C using an ABI 3500XL Genetic Analyser (Applied Biosystems, Foster City, CA, United States) and filter set G5 and POP4 polymers (Applied Biosystems, Foster City, CA, United States). The genotyping data of all samples were collected and analyzed using GeneMapper^®ID-X software (Applied Biosystems, Foster City, CA, United States). Allele peaks were set with an analytical threshold of 50 relative fluorescence unit (RFU).

A total of 33 PCR products AFEC ducks (19), AFEC geese (8), and AFEC Turkeys (6) were sent for sequencing. The number was determined by financial limitations. Sequencing of purified PCR products was performed at Inqaba Biotech, Pretoria, South Africa, using an automated ABI3500XL Genetic Analyser and BigDye terminator v3.1 cycle sequencing reactions (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. DNA data (chromatographs and sequences) were sent back by email for analysis. The same primers used to carry out the amplification of the 16S rRNA gene in the laboratory were used in the sequencing reactions. The sequences were supplied in the form of ab1 files, and the sequence analysis was done using Basic Local Alignment Search Tool in the NCBI databases. Phylogenetic analysis was done using Molecular Evolutionary Genetics Analysis version 7 (MEGA7) [18 ].

HPV positive PCR products either visible in the first or second PCR round were sequenced using respective PCR-product primers with a Big-Dye Xterminator ready reaction kit v3.1 (Applied Biosystems, Foster City, CA. USA) in an ABI 3500xL genetic analyser (Applied Biosystems, Foster City, ca. USA). Genotyping of HPV was inferred by comparing the resulting sequence for each sample to the GenBank database using BLAST (Basic Local Alignment Search Tool). This method identified HPV types in all samples except six.